Water soluble, stabilized peroxidase derivatives, process for their preparation and use in the determination of hydrogen peroxide.

The invention relates to a water-soluble, stabilized peroxidase (POD), a process for its preparation and its use for the determination of hydrogen peroxide.

Peroxidase (EC 1.11.1.7) is an enzyme commonly used in diagnostic reagents. It is used in particular for the determination of hydrogen peroxide, which has arisen in oxidase-catalyzed reactions. This involves an oxidative coupling of color components to a colored product, the amount of which gives a quantitative conclusion about the enzyme or substrate to be determined.

POD is largely stable in buffer solutions, ie the activity does not decrease or only slightly decreases even if it is stored for several days. In the usual reagent compositions for clinical chemistry, which detergents and / or complexing agents, such as. B. EDTA, however, the POD is unstable. Usually, losses of> 90% of the activity are expected in 6 days at room temperature. To ensure that the POD activity is still present to a sufficient extent even after storing such reagents for several days, peroxidase is usually used in a multiple excess.

A major disadvantage of such high POD concentrations, however, is that they result in considerable reagent blank values. This is caused by hem groups which contain the enzyme and which absorb light in the visible range. This leads to inaccuracies in the measurement results.

DE 29 19 622 discloses a process for the preparation of water-soluble enzyme derivatives which are stable in detergent-containing solutions. The enzymes are reacted with polysaccharide derivatives containing aldehyde groups. In contrast to other enzymes modified in this way, which can be stabilized in this way, peroxidase modified in this way does not, however, show sufficiently improved stability in solutions containing detergent and / or complexing agents.

The object of the present invention is therefore to provide water-soluble peroxidase derivatives, the activity of which is retained over a long period of time in aqueous detergent and / or complexing agent-containing solutions.

This object is achieved according to the invention by a water-soluble peroxidase derivative which can be obtained by oxidizing the peroxidase with periodic acid or one of its salts and binding to a water-soluble polymer from the group consisting of polysaccharide, polyethylene glycol, polyvinylpyrrolidone and polyacid anhydride.

Surprisingly, it has been shown that POD derivatives modified in this way show excellent long-term stability in solutions containing detergent and / or complexing agents, without losing essential enzyme properties.

The oxidation of the peroxidase takes place under the conditions familiar to the person skilled in the art with periodic acid or one of its salts, in particular with alkali salts. This is described, for example, in the course of the production of peroxidase-antibody conjugates (E. ISHIKAWA, J. Immunoassay<u style="single">4</u>, 2O9-327 (1983)). Periodic acid or periodate are preferably used in excess. A 20 to 200-fold excess is particularly preferred. The temperature and pH can vary within a wide range. However, it has been shown that the reaction proceeds with particularly good yields in weakly acidic (pH 5-6) and with cooling (0 ° 10 ° C). After the oxidation has been carried out, it is advantageous to remove the oxidizing agent by dialysis or a chromatography step (e.g. on Sephadex® G-25).

In a further step, the oxidized enzyme is bound to a water-soluble polymer. The coupling with this polymer is carried out in a manner known per se by reacting the oxidized POD in aqueous solution with the dissolved polymer under defined conditions (cf., for example, EP 0 069 379).

Suitable polymers are water-soluble polysaccharides, polyethylene glycols, polyvinylpyrrolidones or polyacid anhydrides. A preferred polysaccharide is dextran. However, very good results are also achieved with other water-soluble polysaccharides, in particular with soluble starch. High molecular weight polymers of mono- and disaccharides, such as those obtained by reaction with epihalohydrin (e.g. Ficoll®), have also proven to be very suitable. A copolymer of methyl vinyl ether with maleic anhydride has proven to be particularly suitable as the polyacid anhydride.

The oxidized POD can be bound to the polymer without further intermediate steps, provided that the polymer has reactive groups (e.g. amino groups) which react with the oxidized POD. If the polymer does not have any reactive groups, it is advantageously activated before conversion with the oxidized POD and provided with reactive groups. Methods of this type are familiar to the person skilled in the art and are described, for example, in EP 0 069 379 or in JK Inman, J. Immunol.<u style="single">114</u>, 704 (1975).

The reaction with cyanogen bromide, 1-cyano-4-dimethylaminopyridinium salt (CDAP) or 2,4,6-trichloro-1,3,5-triazine (TCT) proved to be advantageous for the activation of polysaccharides. The reaction with TCT is particularly suitable for activating polyethylene glycol. Likewise, polymers loaded with carboxyl groups can be reacted with N-hydroxy succinimide for activation.

In a preferred embodiment, the oxidized POD is bound to the polymer via one or more alkylene radicals having 1 to 8 carbon atoms in the molecule. For this purpose, the oxidized POD is reacted with the corresponding alkylenediamine and then bound to the optionally activated polymer. Ethylene diamine is particularly preferred as the alkylene diamine.

To stabilize the bond with the alkylenediamine, it is advantageous to connect a reduction step, preferably with borohydride or cyanoborohydride, before reaction with the polymer and, if appropriate, to purify the modified enzyme via a dialysis or chromatography step.

The coupling is advantageously carried out at temperatures between 0 ° and room temperature and pH values ​​between 6 and 10. The molar ratio of enzyme to polymer can be varied within a wide range (1: 1 - 1: 2O). With a molar ratio of approx. 1: 4, however, a particularly high yield of coupling product is obtained.

The POD derivatives according to the invention can be isolated after the coupling reaction has ended, for example by adding acetone and precipitating. However, the solution obtained is preferably lyophilized, expediently after prior purification by dialysis or chromatography.

The derivatization of the POD has no adverse effect on the specificity and activity of the enzyme. On the contrary, it was surprisingly observed that the Michaelis constant (substrate hydrogen peroxide) of the POD derivatives according to the invention is significantly smaller than that of the non-derivatized POD. The derivatives can advantageously be used for the determination of hydrogen peroxide, for example in reagents for clinical-chemical determinations (e.g. uric acid). Due to the smaller Michaelis constant and the improved stability in reagent solutions containing detergent and / or complexing agent, the content of POD derivatives according to the invention can be significantly lower than when using non-derivatized POD.

The following examples further illustrate the invention.

Example 1:

Binding of activated peroxidase to dextran

a) Activation of the POD

5 g horseradish peroxidase (EC 1.11.1.7) are dissolved in 25O ml acetate buffer (30 mmol / l, pH 5.5), 37.5 ml NaJO₄ (0.2 mol / l) are added and 40 min. incubated at room temperature. Then 37.5 ml of 1 mol / l ethylene glycol are added and a further 20 min. incubated at room temperature. The reaction mixture is then purified on a Sephadex G-25 column (mobile solvent: sodium acetate buffer pH 5.5, 10 mmol / l).

b) reaction with ethylenediamine

The pH of the eluate is adjusted to 9.5 with 1 mol / l sodium carbonate / sodium bicarbonate buffer, pH 9.8. Then 11.25 ml of 1 mol / l ethylenediamine solution, pH 9.5, are added. The mixture is then incubated at 22 ° C. for one hour, the pH being kept at 9.5 with the carbonate / bicarbonate buffer mentioned above (pH stat).

The batch is made with 1 mol / l TRA buffer¹⁾, pH 7, O, adjusted to pH 8, O. Then 75O mg of sodium cyano-borohydride are added and incubated for 15 minutes at 22 ° C., the pH value being kept at pH 8.0 with TRA buffer (pH stat). The reaction mixture is then purified on a Sephadex G-25 column (mobile solvent: 10 mmol / l phosphate buffer pH 7, O).

¹⁾(TRA: triethanolamine)

c) binding to dextran

The oxidized POD obtained according to b) is activated on CDAP-BF₄-activated dextran T 4O according to the American Chemical Society Symposium Series in JJ Marshall, Preservation of Enzymes by Conjugation with Dextran <u style="single">123</u> (198O) 125-14O and described by J. Kohn and M. Wilchek, The Use of Cyanogen Bromide and other novel Cyanylating Agents for the Activation of Polysaccharide Resins, Appl. Biochem. Biotech.<u style="single">9</u> (1984) 285-330 modified methods. A specific POD activity of 15-18 U / mg is obtained.

Example 2:

Binding of peroxidase to Ficoll®

As described in Example 1, peroxidase is derivatized and reacted with Ficoll® instead of Dextran T 4O. The yield is: 25-330 g of lyophilisate with a specific POD activity of 14-17 U / mg.

Example 3:

Binding to aminodextran

The reaction mixture prepared according to Example 1b) is treated with 25 g aminodextran (manufactured according to JK Inman, J. Immunol. <u style="single">114</u>, 704 (1975)) (molecular weight <img file="EP0223221A2_D0001.tif" /> 4OOOO) and incubated for 2 hours at room temperature and pH 9.3. Then, as in Example 1b, it is reduced at pH 8.0 with 75O mg of sodium borohydride and, as described in Example 1, purified. The yield obtained is 25-330 g of lyophilisate with a POD activity of 9-14 U / mg.

Example 4:

Binding of native POD to dextran

Horseradish peroxidase is, as described in Example 1c, bound directly to CDAP-BF₄-activated dextran T 4O.

Example 5:

Comparison of the stability of different POD derivatives

<tables id="tabl0001" num="0001"><img file="EP0223221A2_D0002.tif" /></tables>

Composition of reagent a:

0.1 mol / l k-phosphate buffer (pH 8.2) O, 1% NaN₃ 0.1% EDTA 0.5% Lutensol ON 5O 7 mmol / l Na cholate

It can be seen that the stability of non-derivatized (native) POD and of POD fixed without derivatization (Example 4) is only low, while the stability of POD, which was fixed after dextran oxidation, is significantly improved.

Example 6

The stability of POD was examined depending on the detergent and EDTA addition. The following results (incubation time 16 hours at 37 ° C):<tables id="tabl0002" num="0002"><img file="EP0223221A2_D0003.tif" /></tables>

Example 7

The Michaelis constant obtained (substrate hydrogen peroxide) according to the Lineweaver-Burk method in the reagent mix according to Example 5:<tables id="tabl0003" num="0003"><img file="EP0223221A2_D0004.tif" /></tables>